Strategies of Biofilm Disruption

WU Yuqian Supervised by Prof. Margaret Ip 20151215 Department of Microbiology School of Medicine The Chinese University of Hong Kong

Outline Brief Introduction to biofilm D-amino acid treatment Anti-biofilm peptide Drugs targeting oscillation

What is biofilm?

Microorganisms stick on a surface with proteins and polysaccarides

Antibiotics resistance: 10~1000 times Matured biofilm: release planktonic bacteria

Chopra L, et al. (2015) Scientific Reports 5:13412.

D-amino acid treatment

Introduction of D-Amino Acid

Amino acids have L and D forms (except glycine) D-leucine, D-methionine, D-tyrosine and D-

tryptophan Serve as disruption signal, first found in Bacillus

Subtilis (3 days old biofilm; detection after 12 hours of treatment)

D-tyrosine: most effective Disrupting biofilm by releasing TasA fiber

Ilana KG, et al. (2010) Science 328(5978).

Effects of D-Amino Acid

D-tyrosine is most effective on both preventing biofilm formation and disrupting existing biofilm by incorporation into the cell wall

It also inhibits biofilm formation of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa biofilm

Anti-biofilm peptide

Target of Anti-biofilm Peptide

Peptide: immunomodulatory peptide IDR (innate defense

regulator): 1018 (VRLIVAVRIWRR-NH2) Target: guanosine 5'-di(tri)phosphate 3'-diphosphate

[(p)ppGpp] (signal activating stringent response as second messenger)

Principle: inhibition of synthesis of (p)ppGpp and stress response through enzymes RelA and SpoT

de la Fuente-Núñez C, et al. (2015) PLoS One 10(7).

Affectivity of Anti-biofilm Peptide

Material: 8 bacteria (including P. aeruginosa, S. aureus and E. coli)

Detection: flow cytometery (size: syro-9 [green]; death: propidium iodide [red])

Inhibit biofilm growth, eradicate formed biofilm (3-day old); not affect planktonic growth

Low conc. (0.8mg/l) leads to biofilm dispersion; high conc. (10mg/l) leads to biofilm cell death

Overproduction (induced by SHX/IPTG) of (p)ppGpp prevents peptide effect

de la Fuente-Núñez C, et al. (2014) PLoS Pathogens 10(5).

Effects of Anti-biofilm Peptide

Inhibit growth of biofilm Eradicate existing biofilm Increase affectivity of disinfectants (e.g. chlorhexidine)

Oscillation: Introduction and Inspiration

Introduction of Oscillation

Biofilm growth has oscillations (pauses growth periodically)

Biofilm periphery grow → absorb nutrient → biofilm interior starve → (how?) biofilm periphery pause growth → nutrient access biofilm interior → (how?) biofilm periphery grow →→ (oscillation repeats)

Hypothesis: nutrient (e.g. glutamate) triggers metabolic feedback/ion channel communication

Note: glutamate –GDH→ ammonium + glutamine

Hypothesis 1: Ammonium, the signal?

Hypothesis 1: ammonium is a signal to trigger biofilm periphery growth

Result 1: Adding ammonium → oscillation stops, biofilm size continuously increases (microscope)

Result 2: GDH overexpression → oscillation stops, biofilm interior starves and dies (Sytox green dye)

Finding 1: Stress (e.g. H2O2) might promote biofilm growth (though lead to periphery death)

Finding 2: Oscillation pause leads to biofilm interior starve, which benefits periphery elimination

Süel GM, et al. (2015) Nature 523(7562).

Süel GM, et al. (2015) Nature 527(7576).

Hypothesis 2: Ion channel, the signal?

Hypothesis 2: nutrient limitation signal is transported by potassium ion channel

Result 1: biofilm oscillation, membrane potential (thioflavin T) and extracellular potassium (APG-4) changes synchronously

Result 2: high environment potassium charge (e.g. 300mM KCl) pauses membrane potential oscillation

Result 3: extracellular potassium conc. distribution is different from theoretically passive diffusion

Finding 1: active signaling of potassium exist, might take place in oscillation

Hypothesis 2: Ion channel, the signal?

Result 4: existence of channel (yugO) and its gate domain (trkA) [gate opens when glutamate limits, leads to potassium efflux]

Result 5: removal of trkA domain leads to membrane potential constancy loss

Finding 2: potassium ion channel yugO transports glutamate limitation, leads to oscillation

Süel GM, et al. (2015) Nature 527(7576).

Discussion of Oscillation Biocides that lead to death might lead to growth;

signals that lead to growth might lead to death Oscillations of biofilm growth showed that inhibiting growth

of colony periphery might lead to growth of the biofilm interior, vice versa

To eliminate biofilm growth, metabolic products (e.g. ammonium) could be utilized at the same time with disinfectants (e.g. H2O2)

Drugs targeting potassium channel could be utilized by interfering oscillation and metabolic coordination

Not only structural similarities between bacterial and human potassium ion channels, but also their possible functional similarities with respect to electrical communication should be noticed

Conclusion

New strategies have been updated for biofilm growth inhibition and disruption

New methods could be developed according to these strategies

Thank you! Q&A Session

Target of Anti-biofilm Peptide Name Target Reference

Cationic antimicrobial peptides

lipopolysaccharides and lipoteichoic acids

Han HM, et al. (2015) Amino Acids

Cinnamic acid derivative ultrashort tetra-peptide

- Laverty G, et al. (2015) J Pept Sci. 21(10):770-8

Bacteriocins Membrane permeability Chopra L, et al. (2015) Sci Rep. 5:13412.

14-Helical β-peptides - Raman N, et al. (2015) Pharmaceuticals 8(3)

Coryxin (Asn-Arg-Asn-Gln-Pro-Asn-Ser)

- Dalili D, et al. (2015) Colloids Surf B Biointerfaces 135

Peptide 1018 (p)ppGpp de la Fuente-Núñez C, et al. (2015) PLoS One 10(7)

Esculentin-1a(1-21)NH2 Bacterial lipopolysaccharide (LPS)

Di Grazia A, et al. (2015) Amino Acid. 47(12):2505-19

Conclusion

Treatment Advantage Disadvantage

D-Amino Acid Easy to produce, effective Resistance (e.g. yqrM6)

Anti-biofilm Peptide (Peptide 1018)

Could inhibit growth of biofilm, decrease resistance of disinfectant

Resistance (e.g. overexpression of RelA/SpoT)

Metabolic product (e.g. ammonium)

Easy to produce, decrease resistance of disinfectants

Should be utilized together with disinfectant

Drugs targeting ion channels

Low resistance Might need further study

Introduction of Oscillation

500-600μm

Nutrient (e.g. glutamate)

Metabolic products (e.g. ammonium)

1.5-3 hours

Introduction of Oscillation

Growth Rate Nutrient Uptake rate Extracellular potassium

concentration Membrane potential

1.5-3 Hours (Oscillation of Biofilm

Grow

th)

Radius (from interior to periphery)